FR2691546A1 - Particulate tracers for homogeneous immunoassay - comprising antibody, antigen or hapten linked to particulate material without true light absorption - Google Patents

Abstract

Particulate tracers for use in immunoassays comprise an immunoreactive substance (I) and a particulate label (II), (I) is an antibody, antigen or hapten; and (II) is a particle with a max. dimension of at least 0.2 micron comprising a lyophobic or amphilyophilic material having a surface with some (inherent or induced) hydrophilicity and is without true absorption in the visible/near-IR spectrum. The visible/near-IR spectra of the tracers and their aggregates in an aq. medium are the same, irrespective of the particle size attained by antibody-antigen agglutination reaction. Also claimed is a photometric immunoassay method for determn. of an immunoreactive analyte, comprising (a) attaching an antibody, antigen or hapten, by covalent coupling or physical adsorption, to a particulate label whose max. dimension is at least 0.2 micron; (b) contacting the resulting tracer with the analyte in an aq. medium; (c) irradiating the mixt. with light of wavelength 320-1000 nm; and (d) measuring the intensity of the light transmitted by the reaction medium. USE/ADVANTAGE - The method may be used in biological and medical analysis, e.g. for detection of PCR amplification prods. (claimed) or for determn. of serum proteins, drugs such as gentamicin or theophylline, or pathogens such as Toxoplasma gondii. The method is simple, uses stable reagents, and gives detection limits in the ng/ml range with good reproducibility.

Description

DIRECT PARTICULATE TRACTORS, USE
FOR MEASURING AND ASSAYING ANTIBODIES AND
ANTIGENES.

 The present invention as new industrial products relates to direct particulate tracers coupled to any immunoreactive substance; which particulate tracers are characterized mainly by the fact that they have in visible / near infrared light spectral properties similar regardless of their degree of agglutination by antibody / antigen reaction.

 This invention also describes the use of these particulate tracers in an immunological method in a homogeneous phase of qualitative, semiquantitative or quantitative microassay of antibodies and antigens, including haptens.

 By the term direct particulate tracer is meant here the set formed by the association, covalent or passive. of several immunoreactive molecules to the same direct marker in the finely divided state.

 The term direct marker then covers any entity in the particulate state capable of delivering a quantitatively measurable optical signal related to its own structure, and of allowing a covalent or passive surface attachment and the insolubilization of immuno-7active substances. .

 By immuno-reactive substances or molecules is meant all molecules prepared by chemical synthesis or from biological fluids, cellular or tissue extracts obtained in vivo or in Ilirro; especially any monoclonal and polyclonal antibody obtained by cell fusion, or natural or induced immunization, and any antigen, including haptens, such as mediators, peptide and protein hormones, enzymes, structural proteins, respiratory and plasma proteins, nucleoproteins, conjugated proteins, drugs, nucleic acids including PCR amplification products, vitamins, polysaccharides or lipoids.

 By the expression "spectral properties" is meant here the fact that the spectrometric analysis of the particulate tracers according to the invention in the field of visible / near-infrared radiations reveals whatever their degree of agglutination by antibody / antigen reaction. , spectral curves of substantially identical pattern.

 The invention more specifically relates to particulate tracers whose immuno-reactive substance is coupled by covalent chemistry or by simple physical adsorption to a particulate marker whose largest dimension is at least 0.2 micrometer (> m), consisting of a lyophobic or amphilyophilic material, having no true absorption in visible light / near infrared; the use of these particulate tracers in a photometric technique of qualitative, semi-quantitative or quantitative microdosing of any immunologically reactive molecule by reaction of an antibody or an antigen with the corresponding antigen and / or antibody fixed, by covalent chemistry or by simple physical adsorption, to said particulate marker, and, from the measurement in the visible / near infrared wavelength domain, of the light intensity transmitted by the reaction mixture, in proportion to the degree of agglutination tracer after antibody / antigen reaction.

 With the development of laboratory analyzes in both human and veterinary biology, the market for immunodiagnostic tests is very demanding of any new quantitative and semi-quantitative, non-radioactive, rapid, sensitive and simple implementation method. This application primarily concerns the research and measurement of all human and animal biological molecules at low concentrations, eg antibodies, antigens and more generally any molecule naturally or artificially present in an organism or a part thereof, in vivo or In vitro, many sectors, in particular agri-food and toxicological, have opened up to these techniques, and are now in great demand of this type of test, for example for the determination of drugs in body fluids or pyrogens and contaminants in various preparations. .

 The present invention falls within the framework of these uses, in fields as varied as human and veterinary medical diagnosis, biochemical analysis, toxicology, quality control, basic and applied research.

 Among the analytical methods of assay involving an antibody / antigen-type reaction, the most commonly used in immunoassays use tracers, generally of a radioactive, enzymatic, phosphor or particulate nature, which make it possible to objectify the binding. from antigen to antibody.

 The oldest, the radioimmunological techniques (RIA, IRMA) are very competitive by their sensitivity, well below 0.1 nanogram per milliliter (ng.ml-1), and their specificity. Their essential quality is linked to the emission properties of the radioactive marker, unmodified by the physicochemical environment, and to its easy and sensitive detection. However, many constraints of use limit their wide diffusion with the laboratories of analyzes, the principal being related to the very nature of the marker. The possession and use of radiosources are indeed subject to regulation and are only accessible to authorized laboratories, equipped with closed enclosures, dosimetric and radiotoxicological monitoring of personnel, and radioactive waste management. Moreover, the stability of the tracer is only a few months; the implementation of these techniques is not suited to the processing of analyzes piecemeal and requires specific and expensive equipment.

As an alternative to the use of radioactive labels, the coupling of phosphors to antibodies or antigens has been applied to many immunoassay techniques. Sensitivity close to that of radioimmunological methods, these techniques are nevertheless difficult to implement because of limited application areas, the difficulty of accurate measurement of an often fleeting light signal (1 second), maximum fluctuating intensity , numerous washes, an extraction phase of the antibody / antigen complexes. For these reasons,
Process automation and integrated signal measurement are often necessary, requiring complex and often very expensive equipment.

 As for immunoassays using enzyme-labeled tracers, they have grown considerably in recent years. Since they do not require specific equipment, their main advantages are accessibility to all analytical laboratories and sensitivity thresholds of less than 0.1 ng.ml-1. However, these enzymo-immunoassays requiring several reaction stages, immunological then enzymatic, separated from several washing phases, the analysis times are hardly less than two hours and can reach for some molecules 24 to 48 hours. The time and manipulation parameters are the major drawbacks of these techniques, practically prohibiting any piecemeal analysis.

 Finally, there remains the vast field of immunoassays by particle tracer, where the scatterometric properties of insoluble markers in the finely divided state are exploited. Whatever the methodological variations, their principle is all based on the fact that the optical signal produced by the interaction of an electromagnetic wave with the marker is different depending on whether the tracer is free or crosslinked by antibody / antigen agglutination reaction. Among the various approaches to the scatterometer phenomenon, two techniques have emerged in immunoanalysis: turbidimetry (or opacimetry) and nephelemetry, which are differentiated from each other by the optical signal measurement method, respectively by photometry. Absorbance and emission photometry.These techniques are very attractive because of their simplicity and speed, related to the practice of the homogeneous phase reaction, but have a certain number of drawbacks that are not so much related to the principle of reading. implemented, but more to the numerous constraints of exploitation of the laws of diffusion of the light which strongly restrict the fields of use of these techniques (preliminary treatments of the reagents) and the sensitivity of the measurements (10 to 100 ng.ml-1 ). In addition, scatterometric assays may require relatively sophisticated and expensive laser or infrared devices.

 Among the few groups of tracers commonly used in immunoassay, particulate tracers are those that have generated the most work, resulting in multiple methodological variants more or less sensitive and variously applied in immunoassay. But whatever approach is considered, all these techniques come up against the inevitable limitations imposed by the introduction of MIE laws on the diffusion of light by troubled environments.

In this field of particulate tracers, we know in particular the patents
FR-A-2,642,174 and FR-A-2,645,967 which describe latex markers in the submicron particle state (diameter d less than 200 nanometers), the light-scattering properties of which vary according to the degree of d agglutination of the tracer when exposed to electromagnetic radiation in the wavelength range (k) visible / near infrared (300-1000 nm), such as the ratio k / d 2 5: at wavelength The free tracers are not visible photometrically, whereas their aggregates become so after antibody / antigen agglutination. As long as these tracers are used in a turbidimetric or nephelometric process, the sensitivities are respectively at least 100 and 10 ng. ml-1.

 Patent FR-A-2,362,398 and FR-A-2,417,108 are also known which recommend the use of various particulate markers with a diameter of less than 1.6 μm (preferably between 0.2 and 0 μm). 8 μm) in a turbidimetric technique in infrared light (600-2400 nm). Again the process parameters are adjusted so that at the measuring wavelength the free tracer does not absorb light when irradiated with radiation (k) such as X / d 2 1.5. On the other hand, the agglutinates of these tracers at the same wavelength cause an extinction of the light beam transmitted by the reaction mixture. The sensitivity of this technique is of the order of 10-20 ng.ml-1.

A slightly different approach is that of patents FR-A-2,604,526 and FR
A-2,651,326 which consists not in following the appearance of the optical signal specific to the aggregates, but in measuring the decrease in absorbance of the reaction medium at the wavelength corresponding to the maximum true absorption of the free tracer. A-2,651,326, p. 2, lines 1-5]. In this technique, it is very surprising to note that particulate markers consisting of aromatic polymers (polystyrene, polyvinyltoluene, styrene / butadiene) or ethylenic polymers (polyvinylbutadiene) exhibit a maximum true absorption between 320 and 800 nm [FR-A- 2,604,526, p. 19, lines 20-32; Figure 2], while it is perfectly recognized that such conjugated systems have a true absorption between 200 and 300 nm.It is also the fact that these macromolecular markers have no true absorption in the visible spectrum that justifies their wide use in many optical calibration techniques. It is even more surprising to note that for equal constituent material, for example polystyrene, it is only the characteristic envelope (dimensions and shapes) of the tracer (FR-A-2 651 326, p. 8, lines 1-6; p. 9, lines 10-12] which conditions the wavelength of maximum of true absorbance [FR-A-2,651,326, claim 8] whereas by definition the appearance of a true absorption spectrum is independent of the concentration and macromolecular organization of the chromophore.In fact the observations described in these two patents are closer to the scatterometric principles than any other phenomenon, and explain in particular the limits of sensitivity (10 ng.ml-1) and the problems encountered, similar to those of scatterometry.

In general, scatterometric techniques with particulate tracer, turbidimetry and nephelemetry in particular, are limited by several factors:
1. Obligation to select markers with a diameter of less than 0.2 μm for
techniques in visible light and 0.8, um in infrared. This to prevent that,
at the measurement wavelength, the optical background noise due to the free tracer
either too important, decreasing the dynamics of the measured signal by the same amount;
2. obligation to work generally with significant dilutions of tracer
to avoid interference of optical signals between broadcasting centers
adjacent;
3. lack of sensitivity (10-100 ng.ml-1), related to the use of small markers
diameter and low concentrations of tracer.It then takes samples
sufficiently concentrated in analyte to develop optically aggregates
active at the measurement wavelength; aggregates whose growth is of any
limited by the initial dilution of the free tracer;
4. lack of reproducibility, related to instability of pending markers
too diluted (nonspecific tracer agglutination factor) and difficult control
agglutination even under excessively standardized conditions;
5. possible treatment of reagents before dosing; and
6. Implementation sometimes requiring specific and expensive devices.

Therefore, the essential object of the present invention is to exploit novel optical principles that overcome the disadvantages inherent in the simple diffusion laws (MIE laws) involved in turbidimetric, nephelometric and derivative immunoassay methods. by proposing a technical solution based on particulate tracers combining an immuno-reactive substance and a marker in the particulate state, usable in the field of photometric immunoassays, such as ::
1. said immunoreactive substance is selected from antibodies or
antigens, including haptens;
2. said marker is a particle whose largest dimension is at least
0,2 Fm, while dimensions between 0,5 and 4,0 m are
preferable;
3. said marker is made of a material that does not exhibit absorption
true in the field of visible / near infrared wavelengths;
4. said marker is made of a lyophobic or amphilyophilic material and
present on the surface, in a constitutive or induced way, a certain character
hydrophilic necessary for the stability of the tracer in aqueous liquid phase; and
5. the traces of the spectral curves in visible / near infrared light of the said
particulate tracers and their aggregates in aqueous liquid phase are
substantially the same regardless of the dimensions developed by
antibody / antigen agglutination reaction.

 According to another aspect of the invention, a simple, inexpensive and novel homogeneous immunoassay method is carried out using only the minimum of reagent necessary for antibody / antigen agglutination and which implements the original optical properties of the antibodies. particulate tracers mentioned above for the quantitative screening of antibodies and antigens, including haptens, in various test liquids.

 The third object of the present invention is to provide a microdosing process sufficiently simple to implement to be easily adapted to most instruments for photometric measurement, used in the field of biological and medical analysis, and more particularly those allowing the dosing and the partially or totally automated analysis of a multiplicity of samples, so as to minimize the handling and reading times of the measurement.

 Another object of the present invention is to provide a sufficiently sensitive immunological microassay method, on the order of nanograms per milliliter, for detecting extremely small amounts of antibodies and antigens, in all cases where the large sensitivity of radioimmunoassay (RIA) and enzymo-immunological (ELISA) is not required; this with a huge saving of time compared to the RIA and the ELISA (no washing, no phase of extraction of the antibody / antigen complexes, reduced incubation times) and without the biological risks related to the manipulation of radiosources ( RIA).

 Finally, the last aim of this invention is to propose, according to the type of molecule to be investigated, a polyvalent method allowing quantitative or semi-quantitative assays of antibodies and antigens, including haptens, both in the form of direct tests of detection (agglutination reaction) than indirect detection tests (agglutination inhibition reaction). By extension, this method can also be used for qualitative measurements by simply comparing the measured value with a control value, which is significant for the presence of the antibody and / or the antigen to be tested (in screening, for example).

The manner in which the technical solution and the method according to the invention can be realized and the other objects and advantages which result therefrom will emerge more clearly from the discussion and the following detailed examples, in support of the appended figures in which:
FIG. 1 is a diagram illustrating the MIE laws by comparing the apparent spectrophotometric curves obtained for aqueous suspensions of particulate tracers (with a latex marker), the largest dimensions of which are respectively 0.1 μm (Curve 1A), 0.3 llm (Curve 1B), 0.8 Zm (Curve 1C) and 2.0 Fm (Curve 1D).

 FIG. 2 is a diagram illustrating the apparent spectrophotometric curves of different agglutination levels, by antibody / antigen reaction, of a toxoplasma latex tracer having a mean diameter of 0.88 m during a typical serum titration. Positive Gonorrheal Toxoplasma: Buffer Blank (Curve 2A), Serum diluted 1/16 (Curve 2B), 1/8 (2D Curve), 1/4 (Curve 2F), 1/2 (Curve 2E) and Serum pure (Curve 2C).

 FIG. 3 is a diagram illustrating the apparent spectrophotometric curves of different agglutination levels, by antibody / antigen reaction, of a myoglobin latex tracer having a mean diameter of 0.52 Fm during a standard assay of standard solutions of human myoglobin titrated at 25 ng.ml-1 (Curve 3A), 50 ng.ml-1 (Curve 3B), 125 ng.ml-1 (Curve 3C), 250 ng.ml-1 (3D Curve) and 550 ng .ml-1 (Curve 3E).

 Figure 4 shows optical density standard curves measured at wavelengths of 405 nm (Curve 4A) and 540 nm (Curve 4B) using firstly a latex tracer myoglobin having a mean diameter of 0.52, um, and on the other hand different standard solutions of human myoglobin.

 FIG. 5 shows optical density standard curves measured at wavelengths of 405 nm (SA curve) and 540 nm (Curve 5B) using, on the one hand, a kaolin gentamicin tracer having a diameter varying between 1.0 and 4.0 sum, and on the other hand different standard solutions of gentamicin sulfate.

According to the research which led to the development of the present invention, it has been discovered that the optical properties of particulate tracers combining an immunoreactive substance and a marker in the particulate state, characterized in that:
1. said immunoreactive substance is selected from antibodies or
antigens, including haptens;
2. said marker is a particle whose largest dimension is at least
0.2 Clam;
3. said marker is made of a material that does not exhibit absorption
true in the field of visible / near infrared wavelengths; and
4. said marker is made of a lyophobic or amphilyophilic material and
present on the surface, in a constitutive or induced way, a certain character
hydrophilic; no longer followed the laws of Mie; the aforementioned tracers then advantageously having in visible / near infrared light spectral curves of substantially identical pattern, regardless of their degree of agglutination by antibody / antigen reaction.

Until now, particulate tracers used in immunoanalysis had an optical behavior described by the laws of classical diffusion, as illustrated
Figure 1; that is, the intensity diffused at long wavelengths (red radiations) increases with the size of the tracers while it decreases at the small ones (blue radiations). It is this phenomenon which has been clumsily interpreted as a true absorption in FR-A-2 604 526 patents [Figure 3, plate 2/4] and
FR-A-2 651 326. It is also this phenomenon which is hidden behind the terms visible and invisible used in the patents FR-A-2 645 967 and FR-A-2 642 174, and more generally behind all the techniques In practice, we use a measurement wavelength (X) such that the free tracers are not photometrically visible while their aggregates are. To avoid an excessive overlap of the scattering spectra free tracers on the one hand and their aggregates on the other hand - factor of loss of optical signal dynamics and consequently of sensitivity - it is necessary either to be confined to using only markers whose largest dimension (d) is less than 0.2 CLm when working in visible light, or use infrared optical instruments to be able to use markers of larger size (in any case less than 0.8 ÇIm); this in order to maintain an X / d ratio of at least greater than 2, guaranteeing that, at the measurement wavelength, the optical background noise due to the free tracers is minimum. Or if the tracers are too small, Analyzed enough samples are then required to develop optically active aggregates at the measurement wavelength. Moreover, the specificity of the signal generated is poor given the heterogeneity of the aggregates developed by the antibody / antigen reaction; and this especially as the tracer is small. As a result, turbidimetric techniques in visible light are much less sensitive (100 ng.ml-1) than their infrared equivalents (10-20 ng.ml-1).

The other alternative is not to follow the appearance of the optical signal due to the aggregates as in conventional turbidimetry and nephelemetry, but to observe the extinction of the signal generated by free tracers during antibody / antigen agglutination; such as advocates for example patents FR-A-2 604 526 and
FR-A-2 651 326. This is an interesting approach a priori since it links the measured optical signal to the only parameter that does not evolve during the agglutination: the size of the free tracer. Because of this better specificity of the signal, this technique in visible light makes it possible to gain in sensitivity with respect to the classical turbidimetry, but remains limited by the fact that it is necessary nevertheless enough concentrated samples in analyte to move the optical signal sufficiently aggregates and avoid interference with the free tracer.

 In accordance with the investigations carried out in the field of matter / light interactions, it has been found that the particulate tracers according to the invention and their aggregates advantageously have similar spectral properties throughout the visible / near infrared spectrum. For example, transmission spectra determined between 200 to 670 nm wavelength, for different antibody / antigen agglutination levels of aqueous liquid suspensions of toxoplasma latex tracers (0.88 μm in diameter) and latex myoglobin (0, 52 μm in diameter) are shown respectively in Figures 2 & 3. From the examination of Figures 2 & 3, it is clear that for a given tracer, the spectral curves (Curves 2A, 2B, 2C, 2D, 2E, 2F; Curves 3A, 3B, 3C, 3D, 3E) in the wavelength range between 320 and 670 nm have substantially the same plot regardless of the level of agglutination of the tracer by antibody / antigen reaction. This represents a new fact not described in the prior art.

 According to a first embodiment of the invention, wavelengths of between 670 and 1000 nm are also usable for photometric measurement provided that a high sensitivity optical instrument is used, whereas such wavelengths are not considered advantageous, their uses imposing a specific infrared material.

 On the other hand, from the examination of FIG. 2, it appears that the region of ultraviolet wavelengths (200-320 nm) is not exploitable according to the invention because of the high parasitic absorption of many of the components constituting the background media of the tracer suspensions and samples (Curves 2A, 2B, 2C, 2D, 2E, 2F), so that practically only the wavelengths in the visible / near infrared range are usable in principle for the photometric measurement of the degree of agglutination of the tracer.

 Another significant advantage of the tracers according to the invention is that the optical signal generated by the matter / light interaction is no longer described in terms of intensity and optical displacement as in scatterometry, but only in intensity. The independence of the signal measured at the wavelength offers the technical solution according to the invention a great adaptability to all optical instruments suitable for visible / near infrared photometry; This is the case for the majority of devices used in immunoanalysis.

 But much more importantly, is the way in which the intensity of the measured optical signal is associated with the variation in the number and size of the tracers during agglutination. In all previously known scatterometric techniques, which are based on the exploitation of the MIE laws, each free aggregate or tracer is assimilated to a diffusing center which, according to its dimensions, exhibits a characteristic maximum absorption (kmax ap) As shown in Figure 1, scattering centers of 0.1 Rm (Curve 1A), 0.3 Fm (Curve lB), 0.8 Fm (Curve 1C) and 2.0, um (Curve 1D) are characterized by different Bmax ap, respectively 226, 244, 389 and 850 nm (not shown). This finding is a major drawback of scatterometric techniques because agglutination, even under extremely standardized conditions, is not very reproducible. In other words, from one test to the next, it is difficult to obtain a constant degree of agglutination of which the average Bmax ap is generally chosen as the measurement wavelength, with the direct consequence of a poor reproducibility of the measurements and an obvious loss of sensitivity. On the other hand, according to the technical solution according to the invention, the relative intensity variations of the optical signal measured between different levels of agglutination of the tracers being substantially identical over the entire visible / near infrared spectrum, reproducibility is greatly improved, lowering the thresholds of sensitivity.

 Finally, the independence of the optical signal measured at the wavelength advantageously eliminates the limitation imposed by the scatterometry in the choice of the size of the marker. In the technical solution according to the invention, the nature of the measured optical signal being defined only by its intensity component, it is then possible to use markers of large size which increases the detectability of the aggregates, and consequently the sensitivity of the measurement. .

 Moreover, the technical solution according to the invention also makes it possible to increase the precision of the measurements by not using either a signal generated by a negative phenomenon (reduction of the transmitted light) as in turbidimetry, but a signal analogous to a signal of emission (increase of transmitted light), more specific; and this on a simple photometer, without the need to resort to any particular measuring apparatus.

 According to the invention, it is essential for the implementation of such optical properties that the largest dimension of the markers is at least 0.2 μm, preferably between 0.5 and 4.0 μm.

 It is also important that the constituent material of the marker does not exhibit true absorption in the visible / near-infrared wavelength domain.

 Regardless of its optical properties, the other major characteristic of the marker must be its capacity to be able to fix by covalent chemistry or by simple physical adsorption the aforementioned immunoreactive substances. Whatever the coupling mode chosen, it is recommended that the material constituting the marker be lyophobic or amphilyophilic.

 In view of the use of these tracers in immunoassay, the agglutination followed by photometry should be specific to the antibody / antigen reaction between the analyte and the immunological partner coupled to the label. However, the lyophobic nature (total or partial) of the markers according to the invention can cause a great instability (flocculation) of the tracer in the aqueous phase, the solvent most commonly used to form the background media tracers and analytes.

Therefore, according to the invention, it is recommended for the preparation of the tracer that at least 10% of the surface of the label is occupied by hydrophilic groups, polar groups or ionized radicals, constitutive or induced, in particular by peptization with a hydrophilic colloid.

 Thus, the liquid / solid interfacial tensions between the aqueous solvent on the one hand and the marker on the other hand are reduced, and the potential barrier of each tracer is increased; This prevents their coalescence (flocculation) and ensures a high stability (several months) to the immunological reagent consisting of the tracer in aqueous liquid suspension.

 According to the invention, said hydrophilic groups may be in a nonlimiting manner, of the aldehyde (-CHO), aliphatic amine (-CH2-NH2), amide (-CO-NH2), aromatic amine (-4> -NH2) carboxylic acid (-COOH), chloromethyl (-CH2-Cl), hydrazide (NH-NH2), hydroxyl (-OH), thiol (-SH), sulfate (-SO4) or finally sulfonate (-SO3).

 Thus, the typical suitable particulate materials useful for making tracers according to the invention are organic polymers such as polystyrene, polyethylene, polyaminostyrene, polyethylmethacrylate, polypropylene, polyvinylbenzene chloride, polycarboxyvinyl and polyvinyltoluene latices. polymethyl methacrylate, polyacrylic, polyacrylates, styrene / butadiene, styrene / divinylbenzene, styrene / vinyltoluene, styrene / acrylic acid and the like; metallic substances such as gold, silver and the like; mineral substances such as silica, alumina, alumina silicates and the like.

 The new technical solution according to the invention, which is based in particular on the similarity of the spectral properties of free or crosslinked tracers by antibody / antigen agglutination reaction, differs from the teaching of all previously known techniques.

It is thus different from the patents FR-A-2 362 398, FR-A-2 417 108,
FR-A-2,645,967 and FR-A-2,642,174 based on the properties of invisibility / visibility of particulate tracers according to their degree of agglutination, in that the tracers exhibit different spectral properties as a function of their dimensions. In particular, it differs from patents FR-A-2 362 398 and
FR-A-2,417,108 which provide for the use, in infrared photometric, of tracers with mineral or synthetic markers of a diameter not exceeding 1.6 Clam, in that it is described as a major disadvantage the use of light rays having wavelengths of less than 600 nm for monitoring the agglutination of said tracers.It also differs from patents FR-A-2,645,967 and FR-A2,642,174 which provide for the tracking of the agglutination of latex marker particle tracers by visible light / near infrared photometry, in that it is recognized as essential that the dimensions of the markers do not exceed 200 nm.

The present invention is also different from FR-A-2 604 526 and
FR-A-2,651,326 which are based on ultraviolet / visible photometric monitoring of the decay, during the agglutination reaction, of the optical signal generated by particulate marker-labeled tracers with a diameter of between 0.01. and 5.0 clam, in that said tracers have true absorbance in the ultraviolet / visible wavelength range, and aggregates of said tracers have spectral properties different from those of the free tracers.

 The immunoassay method that is recommended according to the invention is an immunological micro-method in liquid / solid homogeneous phase, for qualitative, semi-quantitative or quantitative determination of natural or artificial solutions of antibodies and / or antigens, characterized in that it consists essentially of: associating and insolubilizing, by covalent chemistry or by simple physical adsorption, an immunoreactive substance chosen from antibodies or antigens, including haptens, with a lyophobic or amphilyophilic particulate marker, not showing true absorption for visible / near infrared light and the largest dimension of which is at least 0.2 Clam; contacting the immunological reagent consisting of said tracer in suspension in an aqueous liquid medium with the analyte of the sample to be assayed; and irradiating the reaction mixture with radiation having a wavelength between 320 and 1000 nm, for measuring the intensity of the luminous flux transmitted by said reaction medium.

 The measurement of the variations in luminous flux that is used according to the invention corresponds in fact to the determination of the optical density (OD) or the transmittance (T) of the said reaction media, as can be achieved on any photometer.

 The reason for such an efficiency of the process according to the invention lies in the fact that there is therefore a direct correlation between the degree of agglutination of the tracers of the reaction system and the intensity of the optical signal measured; it also appears that the degree of antibody / antigen agglutination of the tracers is inversely proportional to the logarithm of the analyte, antibody and / or antigen concentration, including the haptens. As is clear from the examination of FIGS. 4 & 5, when a tracer according to the invention is used to photometrically follow the antibody / antigen agglutination, a good correlation is established between the optical density of the reaction mixture and the analyte concentration (Curves 4A, 4B, Curves 5A, 5B), and this regardless of the measurement wavelength in the field of visible / near infrared radiation.

 According to the process according to the invention, it is possible to follow by photometry either the actual agglutination (direct test) or the inhibition of agglutination (indirect test) of the tracers.

 Among the test liquids, plasma, serum, lymph, urine, saliva, cerebrospinal fluid, cell culture supernatants and cell or tissue extracts can be assayed according to the invention. .

 On the other hand, it is considered preferable that the temperature at which the antibody / antigen agglutination reaction is practiced be between 10 and 40 ° C, while a temperature range of 20-30 ° C is more particularly recommended.

 The antibody / antigen agglutination reaction carried out according to the invention can be carried out directly by contacting the sample to be assayed, optionally diluted in an appropriate aqueous solution, with the immunological reagent constituted by the tracer in aqueous liquid suspension. Nevertheless, in order to achieve, in accordance with the above-mentioned objectives, a very sensitive determination of minute amounts of antibodies or antigens in any liquid, it is recommended to promote the contacting of the immunological partners, to be able to employ for at least part of the reaction antibody / antigen a high density of tracer without it leads to its aspecific agglutination, by weak interactions (hydrogen type bonds, ionic, hydrophobic, Van der Waals) between markers or proteins fixed on them.

In this sense, the present invention is also different from the methods of the prior art, which provide a procedure where the agglutination reaction is carried out in a single step (stationary or otherwise), in that, according to the invention, said reaction is carried out with swirling agitation in two stages:
1. The first phase consists of initiating the antibody / antigen reaction by
in contact with the analyte with the tracer at a final concentration of between 0.1
and 2.0% (w / v), to increase the statistical frequency of effective shocks
prevailing in the specific agglutination of tracers; and
2. the second phase consists in continuing the immunological reaction after
diluted the initial reaction mixture with a saline solution of a surfactant,
and thus favor the specific agglutination of the tracer by the analyte of the liquid
test; after which, the intensity of the light flux transmitted by the final reaction medium in the visible / near-infrared wavelength domain is photometrically measured.

 Alternatively, only the first phase of the antibody / antigen reaction can be performed with stirring.

 In the practice of the invention, such a procedure is particularly advantageous because it makes it possible to increase the concentration of the tracer during the initiation phase of the agglutination reaction; it has thus been found that during this first reaction step at least 80% of the antibody / antigen combinations are carried out in the first 15 seconds. Indeed the statistical frequency of shocks between tracers is proportional to the square of their number and consequently that of the aggregates of the reaction medium. But this number decreases to the cube of the dimensions developed by the aggregates. Thus agglutinates ten times larger than the monomeric tracers which constitute them, are a thousand times fewer and therefore meet a million times less often. In the hypothesis that all the shocks are effective, the growth of the aggregates is not limited only by the frequency of their meeting and therefore the initial tracer concentration of the reaction medium. Thus, the procedure according to the invention advantageously makes it possible to reduce the incubation times necessary for the formation of optically active tracer aggregates and, in any case, to develop aggregates of large dimensions, ensuring good dynamics for the signal. measured optical.

 However, any action that promotes the encounters between tracers within a reaction mixture increases the proportion of nonspecific agglutination in parallel, simply by providing the particles with sufficient kinetic energy to cross the potential barriers that protect each tracer from the approach of a neighbor. The dichotomy of the procedure of the method according to the invention advantageously makes it possible to exclude any aspecific agglutination by hydration of the proteins in solution and lowering of the interfacial tensions solid / liquid, by diluting in a second time the initial reaction medium with a saline solution. a surfactant at a concentration which, if added as early as the initial phase of the agglutination reaction, would inhibit or at least greatly impede the antibody / antigen reaction.Among the surfactants usable in the invention nonionic detergents are preferred because experience shows that they are not competitors of the immunoreactive substances adsorbed on the surface of the particles, in the case of non-covalent attachment.

In accordance with the invention, typical suitable surfactants are TRITON
X-100 and TWEEN) at final concentrations of between 0.2 and 0.05% (v / v).

 It is possible, of course, to apply the present invention to determine the kinetics of the antibody / antigen agglutination reaction of the tracer by the analyte, by measuring at regular intervals for a given time the intensity of the light flux transmitted by the analyte. the reaction mixture.

 Preferably, the assay method according to the invention will be carried out on a microplate or on a strip. In practice, this support offers the advantage of being very flexible and allows any laboratory equipped with a photometer of the type used for enzymo-innounalysis, to easily treat small as well as large series of samples (8 to 96 simultaneously), with a minimum cost.

 The assay can also be performed on any automatic apparatus of the biochemistry analyzer type, and any spectrophotometer with, in counterpart, a more restrictive use in the latter case, taking into account the greater number of manipulations necessary for the implementation of the process for the treatment of series of samples.

 Given the versatility of implementation of the method according to the invention, a kit for photometric immunoassay comprises at least the two basic reagents (immunological reagent, reaction diluent) necessary for the antibody / antigen reaction under the conditions above. Optionally this kit may include the various accessories specific to the implementation of the method according to the invention on each optical instrument for a photometric measurement.

In the process according to the invention, the combination of the technical novelties related to the originality of the operating mode and the optical properties of the tracers according to the invention advantageously makes it possible to achieve:
1. Sensitivity thresholds in the order of nanograms per milliliter (ng.ml-1),
so that this process is perfectly adapted to the main applications
current immunological
2. very good reproducibility and repeatability of measurements, varying
respectively according to the implementation protocols (manual or automatic)
2.0 to 7.0% and 0.1 to 2.0%;
3. a great simplicity of implementation, without the need for any treatment
preliminary reagents or test liquids;
4. a great adaptability to most instruments allowing a measurement
visible / near infrared photometric; and
5. high stability of the immunological reagent; and overcomes the many disadvantages of previously known scatterometric methods which are imprecise and insensitive, given the low specificity of the measured optical signal, the large number of factors that can interfere with the measurement (dust, any impurities), the use of weak markers diameter and suspensions of very dilute tracers, the instability of the tracers, the fact of generally conducting the reaction in stationary state or finally the difficult control of nonspecific agglutination during very long incubation times.

 The manner in which the process according to the invention can be carried out and the advantages which derive therefrom will emerge more clearly from the nonlimiting examples which follow.

EXAMPLE 1. Preparation of an adsorbed anti-myoglobin latex reagent.

4 ml of a tris (hydroxymethyl) -aminomethane HCl 0.10 molar solution pH = 7.5 of human antimyoglobin antibody (SIGMA, France) are placed in a glass tube at a concentration of 4 milliliters (ml). of 0.7 mg.ml-1, then 0.5 ml of a commercial solution of carboxylated polystyrene latex (0.52 clam) at a concentration of 10% by weight (reference P0005070PN) are added with stirring,
BANGS LABORATORIES, USA). After stirring for 2 minutes, the mixture is allowed to stand for 8 hours at room temperature, then 16 hours at +4 ° C. The mixture is then centrifuged at 10,000 g, 20 minutes, +4 ° C. and the pellet is taken up in a tris buffer. - (hydroxymethyl) -aminomethane HCl 0.1 M pH = 7.5. We thus practice two successive washes. The last pellet is taken up and adjusted to storage buffer (tris (hydroxymethyl) aminomethane buffer 0.10M HCl PU = 7.5, PEG 6000 3.0%, Sodium azide 19.1-1).

Under these conditions of preparation, the adsorbed anti-myoglobin latex reagent is stable for at least 3 months, and exhibits no desorption phenomenon.

EXAMPLE 2 Quantitative determination of serum myoglobin by direct latex test for the diagnosis of muscle and cardiac lesions (infarction)
In the microcupules of a microplate or an ELISA-type strip, 20 microliters (> 1) of different standard solutions of myoglobin (25 50, 125, 250, 550 ng · ml-1 of human myoglobin; SIGMA, France) which is reacted for 1 minute with stirring at room temperature with 10 μl of the anti-myoglobin latex reagent adsorbed as prepared in (1). The reaction volume is then diluted with 100 μl of 0.25% TRITON49 X-100 saline and stirred for 2 minutes.

The optical density (OD) measurement is made at 405 and 540 nm on an ORTHO DIAGNOSTIC SYSTEMS microplate optical reader.

The results recorded in Table A are thus obtained.

When, from the data below, a graph is plotted on the abscissa of the logarithm of human myoglobin concentration and the OD values measured at 405 nm (Curve 4A) and 540 nm (Curve 4B) are plotted on the ordinate. Standard curves thus established have the appearance of substantially straight parallel lines, as shown in Figure 4, at least for the susspecified concentrations of myoglobin.

TABLE A. MYOGLOBIN STANDARD RANGE / LATEX REAGENT

<tb> Concentration
<tb> of <SEP> myoglobin <SEP> D0405 <SEP> nm <SEP> OD540 <SEP> nm
<tb><SEP> 25 <SEP> ng.ml-1 <SEP> 1.128 <SEQ> 0.955
<tb><SEP> 50 <SEP> 1.008 <SEP> 0.864
<tb><SEP> 125 <SEP> 0.855 <SEP> 0.725
<tb><SEP> 250 <SEP> 0.742 <SEP> 0.626
<tb><SEP> 550 <SEP> 0.611 <SEP> 0.514
<Tb>
The OD values obtained for test liquids tested according to the same protocol, are compared with the semi-logarithmic calibration curve established at any of the measurement wavelengths in the range 320-670 nm, This makes it possible to determine the human myoglobin concentration of the assayed samples, with identical sensitivities regardless of the measurement wavelength in the field of visible / near infrared radiation.

EXAMPLE 3 Preparation of a reagent kaolin gentamicin adsorbed.

at. In a glass tube, 40 milligrams (mg) of bovine albumin (BSA, SIGMA, France) and 50 mg of gentamicin sulfate (SIGMA, France) are dissolved in 2 ml of an aqueous solution pH = 6.5 mm. a water-soluble carbodiimide, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (ALDRICH, France), at a concentration of 2 mg.ml-1, prepared extemporaneously. After incubating for 4 hours at 37 ° C. with stirring, the mixture is dialyzed for 12 hours at + 4 ° C. against 0.10 M glycine buffer pH = 8.2.

b. In a glass tube, 4 ml of a 0.10M glycine buffer pH = 8.2 solution of gentamicin / BSA conjugate prepared in (a) at a concentration of 1.0 mg.ml-1 BSA equivalent is placed, 30 mg of kaolin (calibrated hydrated alumina silicate, 1.0-4.0 μm, SIGMA, France) are then added with stirring. After stirring for 2 minutes, the mixture is left to stand for 8 hours at room temperature, then 16 hours at +4 ° C. The mixture is then centrifuged at 6000 g, 30 minutes, +4 ° C. and the residue is taken up in glycine buffer. 10M pH = 8.2 Two successive washes are thus carried out The last pellet is taken up and adjusted in storage buffer (0.10M glycine buffer pH = 8.2, 0.1% BSA, 19.1-1 Sodium azide ).

Under these conditions of preparation, the reagent kaolin gentamicin adsorbed is stable for at least 3 months, and exhibits no desorption phenomenon.

EXAMPLE 4. Quantitative determination of gentamicin by indirect kaolin test (inhibition of agglutination).

In the microcupules of a microplate or an ELISA-type strip, 20 μl of the different points of a liquid standard range (15; 30; 60 120; 240; 480 ng · ml-1 of gentamicin) are deposited in pairs. that is reacted at room temperature with 20 μl rabbit anti-gentamicin / BSA antiserum (SIGMA,
France) diluted to 1/200 in 0.10M glycine buffer pH = 8.2. After stirring for 1 minute, 10 μl of the adsorbed kaolin gentamicin reagent as prepared in (3) are added and this mixture is incubated under the same stirring conditions for 2 minutes. The reaction volume is then diluted with 150 Cr of 0.2% TRITONS X-100 saline and stirred for 2 minutes.

The OD measurement is made at 405 and 540 nm on an optical microplate reader
ORTHO DIAGNOSTIC SYSTEMS.

The results recorded in Table B below are thus obtained.

TABLE B. GENTAMICINE / KAOLIN REAGENT STANDARD RANGE

<tb><SEP> Concentration <SEP>
<tb> of <SEP> gentamicin <SEP> OD405 <SEP> nm <SEP> OD0 <SEP> nm
<tb><SEP> 15 <SEP> ng.ml-l <SEP> 0.735 <SEP> 0.702
<tb><SEP> 30 <SEP> 0.638 <SEP> 0.601
<tb><SEP> 60 <SEP> 0.551 <SEP> 0.511
<tb><SEP> 120 <SEQ> 0.449 <SEP> 0.415
<tb><SEP> 240 <SEP> 0.357 <SEP> 0.316
<tb><SEP> 480 <SEP> 0.276 <SEP> 0.229
<Tb>
When establishing for the wavelengths of 405 nm (Curve 5A) and 540 nm (Curve 5B) the semi-logarithmic standard curves on the basis of the above data, as shown in Figure 5, the existence of a narrow correlation between the gentamicin concentration and the OD value, regardless of the measurement wavelength.

Consequently, the OD values obtained for test liquids tested according to the same protocol, can be compared with one of the semi-logarithmic calibration curves thus established, thus making it possible to determine the gentamicin concentration in n ' any sample.

EXAMPLE 5 Preparation of an Adsorbed Genfamicin Latex Reagent

at. The gentamicin / BSA conjugate is prepared as in (3a), except dialysis which is performed against a 0.15M phosphate buffer pH = 7.8.

b. In a glass tube, 4 ml of a solution in 0.15M phosphate buffer pH = 7.8 of gentamicin / BSA conjugate prepared in (a) at a concentration of 0.8 mg.ml-1 BSA equivalent, then 0.5 ml of a commercial solution of carboxylated polystyrene latex (0.52 μm) at a concentration of 10% by weight (Ref P0005070PN, BANGS LABORATORIES, USA) is added with stirring. After stirring for 2 minutes, the mixture is allowed to stand for 8 hours at room temperature, then 16 hours at +4 ° C. The mixture is then centrifuged at 10,000 g, 20 minutes, +4 ° C. and the pellet taken up in the same buffer. phosphate. We thus practice two successive washes. The last pellet is taken up and adjusted into storage buffer (0.15M phosphate buffer pH = 7.8, PEG 6000 3% Sodium azide 19.1-1).

Under these conditions of preparation, the adsorbed gentamicin latex reagent is stable for at least 3 months, and exhibits no desorption phenomenon.

EXAMPLE 6. Quantitative determination of gentamicin by indirect latex test (inhibition of agglutination).

In the microcupules of a microplate or of an ELISA-type strip, 20 μl of the different points of a liquid standard range (15; 30; 60 120; 240; 480 ng · ml-1 of sodium sulfate) are deposited in pairs. gentamicin) which is reacted at room temperature with 20 μl of rabbit anti-gentamicin / BSA antiserum (SIGMA, France) diluted 1/200 in 0.15M phosphate buffer pH = 7.8. After stirring for 1 minute, 10 μl of the adsorbed gentamicin latex reagent as prepared in (5) are added and this mixture is incubated under the same stirring conditions for 2 minutes. The reaction volume is then diluted with 50 μl of 0.1% TRITON® X-100 saline and stirred for 2 minutes.

The OD measurement is made at 405 and 540 nm on an ORUHO DIAGNOSTIC SYSTEMS microplate optical reader.

The OD values obtained for test liquids tested according to the same protocol, are compared with the semi-logarithmic calibration curve established from the standard range data below (Table C), which makes it possible to determine the gentamicin concentration of the assayed samples.

TABLE C. GENTAMICINE / LATEX STANDARD RANGE

<tb> Concentration
<tb> of <SEP> gentamicin <SEP> DO405nm <SEP> DO540 <SEP> nm
<tb><SEP> 15 <SEP> ng.ml-l <SEP> 1,243 <SEP> 1,123
<tb><SEP> 30 <SEP> 1,108 <SEP> 0.991
<tb><SEP> 60 <SEP> 0.968 <SEP> 0.860
<tb><SEP> 120 <SEP> 0.835 <SEP> 0.735
<tb><SEP> 240 <SEP> 0.698 <SEP> 0.601
<tb><SEP> 480 <SEP> 0.581 <SEQ> 0.478
<Tb>
EXAMPLE 7 Preparation of an adsorbed theophylline latex reagent

at. In a glass tube, 40 mg of hemocyanin (KLH, SIGMA, France) and 50 mg of a carboxylated derivative of theophylline, 8- (3-carboxypropyl) -1,3 dimethylxanthine (SIGMA, France) are dissolved. in 2 ml of an aqueous solution pH = 5.5 of 1 (3-dimethylaminopropyl) -3-ethylcarbodiimide (ALDRICH, France) at a concentration of 2 mg.ml-1, prepared extemporaneously. After 4 hours of incubation at 37 ° C. with stirring, the mixture is dialyzed for 12 hours at + 4 ° C. against 0.15M phosphate buffer pH = 7.8.

b. In a glass tube, 4 ml of a solution in 0.15M phosphate buffer pH = 7.8 of theophylline / KLH conjugate prepared in (a) is placed at a concentration of 0.8 mg.ml-1 BSA equivalent, then 0.5 ml of a commercial solution of carboxylated polystyrene latex (0.52 μm) at a concentration of 10% by weight (Ref P0005070PN, BANGS LABORATORIES, USA) is added with stirring. After stirring for 2 minutes, the mixture is allowed to stand for 8 hours at room temperature, then 16 hours at +4 ° C. The mixture is then centrifuged at 10,000 g, 20 minutes, +4 ° C. and the pellet taken up in the same buffer. phosphate. Two successive washes are thus carried out.The last pellet is taken up and adjusted into storage buffer (0.15M phosphate buffer pH = 7.8, 3% PEG 6000, 19.1-1 Sodium azide).

Under these preparation conditions, the adsorbed theophylline latex reagent is stable for at least 3 months, and exhibits no desorption phenomenon.

EXAMPLE 8 Quantitative determination of theophylline by indirect latex test (inhibition of agglutination).

In the microcupules of a microplate or of an ELISA-type strip, two points of a liquid standard range (7.5, 15, 75, 150 ng.ml -1 of theophylline; SIGMA, France) which is reacted at room temperature with 20 μl of an anti-theophylline / KLH rabbit antiserum (SIGMA, France) diluted 1/300 in 0.15M phosphate buffer pH = 7.8. After stirring for 1 minute, 10 μl of the theophylline latex reagent adsorbed as prepared in (7) is added and this mixture is incubated under the same stirring conditions for 2 minutes. The reaction volume is then diluted with 50 μl of 0.1% TRITON saline (9 X-100) and stirred for 2 minutes.

The OD measurement is made at 405 nm on an ORTHO microplate optical reader
DIAGNOSTIC SYSTEMS.

The OD values obtained for test liquids tested according to the same protocol, are compared with the semi-logarithmic calibration curve established from the standard range data below (Table D), which makes it possible to determine the theophylline concentration of the assayed samples.

TABLE D. THEOPHYLLINE / REACTIVE LATEX RANGE

<tb> Concentratian
<tb> of <SEP> theophylline <SEP> D0405nm
<tb><SEP> 7.5 <SEP> ng.ml-1 <SEP> 1,098
<tb><SEP> 15 <SEP> 0.941
<tb><SEP> 30 <SEP> 0.768
<tb><SEP> 75 <SEP> 0.570
<tb><SEP> 150 <SEP> 0.401
<Tb>
EXAMPLE 9 Preparation of a Covalent TgAg Latex Reagent

at. 10 mg of carboxylated polystyrene latex with a diameter of 0.88 μm are activated for 10 minutes at 50 ° C. (Ref P0008801CN, BANGS
LABORATORIES, USA) washed several times with deionized water, with 1 mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide (ALDRICH, France). The activated latex is centrifuged, washed and redispersed in 0.15M phosphate buffer pH = 7.8. The latex thus activated is to be used within two hours of its preparation.

b. Then added to the aqueous suspension of activated latex prepared in (a) 1 ml of a lysate of Toxoolasma aondii at a concentration of 3 mg.ml-1 equivalent BSA, in 0.15M phosphate buffer pH = 7.8 and Incubate the mixture for 10 minutes at 50 ° C. The sensitized latex is then centrifuged, washed twice in 0.15M phosphate buffer pH = 7.8 and then taken up and adjusted in storage buffer (0.18 pH buffer pH = 7.8 PEG 6000 3% Sodium Azide 19.1-1).

Under these conditions of preparation, the covalent TgAg latex reagent is stable for at least 3 months.

EXAMPLE 10 Semi-quantitative assay by direct latex test of the antibodies
Total TgAg in screening for Toxoolasma aondii infection.

In the microcupules of a microplate or an ELISA-type strip, a control serum containing 30 μl of a negative control serum, a positive control serum adjusted to twice the positivity threshold and serums to be assayed diluted with tenth in 0.1su phosphate buffer pH = 7.8. 10 μl of covalent TgAg latex reagent as prepared in (9) are then added to the same microcupules. After stirring for 1 minute, the reaction volumes are diluted with 100 μl of a 0.2% TRITONS saline solution.
X-100 and stirred for 2 minutes.

The OD measurement is made at 405 nm on an ORTHO microplate optical reader
DIAGNOSTIC SYSTEMS.

The OD values obtained are expressed as the ratio:
Negative control
Sample OD and are listed in Table E below, in comparison with a standard technique, a Toxoplasma ELISA.

TABLE E

<tb> Patient <SEP> ELISA <SEP> Toxo <SEP> Latex <SEP> Toxo
<tb> S12-15 <SEP><<SEP> 1 / Negative <SEP> ~ <SEP> 1 / Negative <SEP>
<tb> S15-121 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S15-179 <SEP> 2.29 / Positive <SEP> 1.47 / Positive
<tb> IF <SEP> 5-17 <SEP> 2.87 / Positive <SEP> 1.35 / Positive
<tb> S1 <SEP> 3-42 <SEP> 7,20 / Positive <SEP> 1,36 / Positive <SEP>
<tb> S16-22 <SEP> 1.43 / Doubtful <SEP> 1.62 / Positive
<tb> If <SEP> 2-17 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S1 <SEP> 5-1 <SEP> 51 <SEP> 5.33 / Positive <SEP> 1.13 / Positive <SEP>
<tb> S12-19 <SEP><<SEP> 1 / Negative <SEP> ~ <SEP> 1 / Negative <SEP>
<tb> S12-81 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S1 <SEP> 5-1 <SEP> 00 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S1 <SEP> 5-1 <SEP> 01 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S15-95 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> 515-18 <SEP> 2.07 / Positive <SEP> 1.43 / Positive
<tb> S23-48 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S23-89 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S23-11 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<tb> S77-15 <SEP><<SEP> 1 / Negative <SEP> - <SEP> 1 / Negative
<Tb>

Claims (23)

 antibody / antigen agglutination reaction.  substantially the same regardless of the dimensions developed by  particulate tracers and their aggregates in aqueous liquid phase are  - the traces of the spectral curves in visible / near infrared light of the said  hydrophilic; and  present on the surface, in a constitutive or induced way, a certain character  said marker consists of a lyophobic or amphilyophilic material and  in the field of visible wavelengths / near infrared;  said marker is made of a material that does not exhibit true absorption  0.2 micrometer;  said marker is a particle whose largest dimension is at least  antigens, including haptens;  said immunoreactive substance is chosen from antibodies or  1. Particulate tracers comprising an immuno-reactive substance and a marker in the particulate state, usable in the field of assays involving an antibody / antigen reaction, characterized in that: 2. particulate tracers according to claim 1, characterized in that the markers are chosen from groups consisting of particles of a synthetic polymer, particles of a mineral substance or particles of a metal. 3. Particulate tracer according to claim 2, characterized in that the synthetic polymer is chosen from homopolymers of styrene, ethylene, vinyl toluene, methyl methacrylate, ethyl methacrylate, propylene, vinyl benzene chloride, vinyl carboxylated, acrylic acid, various acrylates, aminostyrene; among styrene / butadiene, styrene / divinylbenzene, styrene / vinyltoluene, styrene / acrylic acid copolymers. 4. Particulate tracer according to claim 2, characterized in that the mineral substances used are silica, alumina, alumina silicates. 5. Particulate tracer according to claim 2, characterized in that the metals used are gold or silver. 6. Tracers according to claim 1, characterized in that several molecules of said immunoreactive substance are fixed covalently or by simple physical adsorption to each particulate marker. 7. Tracers according to claim 1, characterized in that the largest dimension of the marker is between 0.5 and 4.0 micrometers. 8. Tracer according to claim 1, characterized in that the hydrophilic character is carried by at least 10% of the surface of the marker. 9. Tracer according to claim 1, characterized in that the hydrophilic surface character of the marker is provided by any chemical function of the aldehyde type (-CHO), aliphatic amine (-CH2-NH2), amide (-CO-NH2), aromatic amine (-4> -NH2), carboxylic acid (-CO OH), chloromethyl (-CH2-Cl), hydrazide (NH-NH2), hydroxyl (-OH), thiol (-SH), sulfate (-SO4) or sulfonate (-SO3). 10. Tracer according to claim 1, characterized in that the hydrophilic surface character of the marker is provided by peptization with a hydrophilic colloid. 11. Use of tracers according to any one of claims 1 to 10 in the field of photometric immunoassays.  12. Method for photometric measurement and immunoassay of any immuno-reactive substance, characterized in that it consists essentially of: associating and insolubilizing, by covalent chemistry or by simple physical adsorption, the immuno-reactive substance chosen from antibodies or antigens, including haptens, to a particulate marker the largest dimension of which is at least 0.20 microns; contacting said tracer with the analyte of the sample in an aqueous liquid medium; and irradiating the reaction mixture with a light having a wavelength between 320 and 1000 nanometers, to measure the intensity of the light flux transmitted by the reaction medium. 13. The method of claim 12, characterized in that it implements any means promoting the specific agglutination of the tracer by the analyte. 14. The method of claim 13, characterized in that the agglutination reaction of the tracer by the analyte is conducted with vortex stirring in two phases: the first is to initiate the antibody / antigen reaction by contacting the analyte with the tracer at a final concentration of between 0.1 and 2.0% (w / v); and then to continue the immunological reaction after diluting the initial reaction mixture with a saline solution of a surfactant. 15. The method of claim 14, characterized in that the surfactant is nonionic type. 16. The method of claim 14, characterized in that the surfactant is used in the reaction mixture at a concentration between 0.2 and 0.05% (v / v). 17. The method of claim 12, characterized in that the antibody / antigen agglutination reaction is carried out in the temperature range of 10 to 40 ° C and preferably between 20 and 30 OC. 18. A method according to claim 12, characterized in that the light applied for the photometric measurement is selected in the range of wavelengths between 340 and 620 nanometers. 19. Method according to any one of claims 12 to 18, characterized in that photometry is measured by either agglutination or tracer agglutination inhibition. 20. Process according to any one of claims 12 to 19, characterized in that the kinetics of the antibody / antigen agglutination reaction of the tracer by the analyte are photometrically measured. 21. Method according to any one of claims 12 to 19, characterized in that the intensity of the luminous flux transmitted by the reaction mixture is measured by photometry after a reaction time of a prescribed duration for specific conditions. 22. Application of the method according to any one of claims 12 to 21, for the detection of amplification products by PCR (Polymerase Chain Reaction). 23. Required for photometric immunoassay, characterized in that it comprises the various reagents and accessories necessary for carrying out the process according to claims 12 to 21.